Hormonal treatment of endometrial cancer: past, present and future

Best Practice & Research Clinical Obstetrics and Gynaecology Vol. 15, No. 3, pp. 469±489, 2001

doi:10.1053/beog.2000.0189, available online at http://www.idealibrary.com on

10 Hormonal treatment of endometrial cancer: past, present and future Edward Podczaski

MD

Professor, Division of Gynecologic Oncology and Department of Obstetrics and Gynecology

Rodrigue Mortel

MD

Associate Dean and Director, Penn State Cancer Center, and Professor, Obstetrics and Gynecology Penn State College of Medicine, The Milton S. Hershey Medical Center, 500 University Drive, Hershey, PA 17033-2390, USA

The concept that hormonal therapy may be useful in the treatment of endometrial cancer antedated the pharmaceutical availability of progestational compounds. By 1959, initial studies demonstrated the ability of progestins to reverse endometrial hyperplasias. Thereafter, progestins and other hormonal agents have been used in various roles as treatment for endometrial cancers. This chapter reviews the use of hormonal agents for the treatment of primary and metastatic/recurrent endometrial cancer, as well as such treatment in an adjuvant setting. Major problems in enhancing the ecacy of endocrine therapy of cancers arising from hormonally responsive tissues are also considered. The regulations of steroid-hormone receptor expression in endometrial and breast cancers continues to be an active area of research interest. Key words: endometrial cancer; oestrogen receptor; progesterone receptor; progestins; tamoxifen.

THE PAST Following the initial success of hormonal ablation in the treatment of breast and prostate cancers, sex steroids were considered in the therapy of other cancers derived from hormonally responsive tissues. The profound e€ects of progesterone on the normal endometrium and the inherent hormonal sensitivity of this tissue led to the concept that progestins may be useful in the treatment of endometrial cancer. Unfortunately, trials testing this hypothesis were initially limited by the lack of pharmaceutically potent progestins. By 1959, detailed studies by Kistner demonstrated that synthetic progestins produced marked changes in the glands and stroma in cases of adenomatous hyperplasia and adenocarcinoma in situ of the endometrium.1 Glandular atrophy and decidualization of the stroma were consistent ®ndings following prolonged progestational therapy. Using 17-a hydroxyprogesterone caproate or progesterone in oil, Baker reported objective regression of tumour in ®ve of 15 patients with advanced or recurrent endometrial cancer.2 Factors predicting a favourable response to progestins included a 1521±6934/01/030469‡21 $35.00/00

c 2001 Harcourt Publishers Ltd. *

470 E. Podczaski and R. Mortel

long hiatus between primary therapy and the appearance of metastasis, a histologically well-di€erentiated endometrial adenocarcinoma, and a pulmonary site of metastasis.3 In favourable cases, pulmonary metastases regressed within 2 months of the initiation of therapy and disappeared by 4±6 months. Responsive osteolytic lesions calci®ed, resulting in symptomatic relief of pain and disability. Kennedy administered 17-ahydroxyprogesterone caproate to 27 patients with advanced endometrial cancer.4 Objective improvement occurred in eight patients (29.6%). Adenocarcinomas responsive to progestins were of long duration and slow growth, whereas refractory patients had tumours of short duration and rapid growth. The progestin-induced regression of advanced endometrial cancers was attributed to a direct hormonal e€ect on the tumour. The mechanism of progestin action was clari®ed with the availability of radioactively-labelled sex steroids. Hormonally responsive tissues contain protein receptors which speci®cally bind to the steroid ligand. These receptor proteins demonstrate special characteristics: the binding of the hormonal ligand is speci®c, of high anity and saturable. Furthermore, the binding of hormone to receptor results in a biological response. Early studies indicated that the initial binding of sex hormone with its receptor protein took place in the cytoplasm. The hormone±receptor complex was then `translocated' into the nucleus. The bound complex then interacted with a unique portion of the DNA, resulting in RNA transcription and a speci®c response in target tissues. Ultimately, such interactions allow nanomolar concentrations of a particular hormone to e€ect major alterations in target cell phenotype. In the case of progestin administration, this phenotype results in diminished mitotic activity and the promotion of cellular di€erentiation within the endometrium. At the cellular level, progestin administration results in downregulation of both oestrogen and progesterone receptors. THE PRESENT The modern concept of the sex steroid receptor As a result of early studies with radioactively labelled steroids, the initial binding of oestrogen or progestin with their speci®c receptors was thought to occur within the cytoplasm of hormonally responsive tissues. However, immunohistochemical methods using monoclonal antibodies raised against the oestrogen and progesterone receptor have localized these binding proteins exclusively to the nuclei of target cells (Figure 1). As such, cytosolic assays of oestrogen and progesterone receptor concentrations actually measure nuclear receptor released during tissue processing. Recombinant DNA techniques have permitted the study of the gene sequences encoding the progesterone and oestrogen receptors. These receptors share a common structure with the receptors for thyroid hormone, androgens, adrenal steroids, 1,25-dihydroxy vitamin D3 , and retinoic acid.5,6 These nuclear receptors form a superfamily of more than 100 proteins present in practically all species from invertebrates to humans. In addition, this family also includes a large number of receptors with, as yet, unidenti®ed ligands (orphan receptors). Each nuclear receptor contains characteristic domains that are similar, regardless of the hormonal ligand. Analysis of the genes encoding the superfamily receptors suggests a complex evolutionary history during which gene duplication events and swapping between domains of di€erent origins took place.7 Members of the superfamily of nuclear receptors have a modular structure with conserved domains A through F (Figure 2).8 The A/B region at the amino-terminal end

Hormonal treatment 471

Figure 1. Immunohistochemical localization of the progesterone receptor (PR) in sections of normal endometrium. Staining for PR is limited to the nuclei of glandular and stromal cells. Courtesy of Dr R. Zaino, Department of Pathology, Penn State University School of Medicine, Hershey Medical Center, Hershey, PA, USA.

DNA Binding Domain NH2

A/B

Regulatory Domain

C

Hormone Binding Domain D

E

F

COOH

Hinge

Figure 2. Domain structure of members from the superfamily of nuclear receptors.

of receptor proteins demonstrates the greatest variability in size and sequence. In contrast, the C and E domains are the most highly conserved, suggesting that these regions are important for common receptor function. The receptor proteins contain domains for DNA binding (C domain), hormone binding and dimerization (E domain), as well as transcriptional activation. The region between the DNA-binding and hormone-binding domains contains a signal area responsible for the nuclear compartmentalization of the receptor following its synthesis in the cytoplasm. This region is also a site of rotation as a result of conformational changes in the receptor, hence the name hinge (or D) domain. The overall mechanism of action for the oestrogen and progesterone receptor is similar (Figure 3). In the absence of hormone, the transcriptionally inactive receptor is located in the nuclei of target cells as a large macromolecular complex associated with several heat shock proteins.9 Upon interaction with its respective hormonal ligand, the receptor undergoes conformational changes permitting disassociation of the inhibitory proteins. The hormone-bound receptor then spontaneously dimerizes and acquires

472 E. Podczaski and R. Mortel

1. Binding of Hormone to its Receptor (R)

+

+

Hormone Heat Shock Proteins -R Complex

Heat Shock Proteins

2. Dimerization of Bound Receptors

+ Dimerized Bound Receptors

3. Binding of Dimer to HRE

4. Formation of Transcriptional Machinery And mRNA from Target Genes

HRE

Adaptor Proteins Cofactors

DNA

DNA Target Genes

RNA Polymerase

Messenger RNA

Figure 3. Classical model of oestrogen receptor action. Binding of hormone to the receptor results in displacement of heat shock proteins (1), receptor dimerization (2) and eventual DNA binding at the oestrogen-responsive element (3). Assembly of the transcriptional machinery results in the formation of mRNA transcribing target genes located downstream of the hormone-responsive element (4).

the ability to bind strongly to a speci®c DNA site (the hormone responsive element ± HRE) by means of the zinc ®nger area of the DNA-binding domain. Steroid hormone receptor controls gene expression through binding, as dimers, to short palindromic hormone-responsive elements located upstream of the genes they regulate.10,11 The progesterone receptor The human progesterone receptor appears in two sizes: the high-molecular-weight (B) form, containing 933 amino acids, and a low-molecular-weight (A) form lacking a 164 amino acid fragment from the amino-terminal of the B isoform.9 Both forms of the receptor are derived from a single gene as a consequence of alternative initiation of transcription from distinct promoters. Progestational agents can produce a variety of responses in target tissues as determined by the receptor expressed (A or B) and the activity of the dimers produced (homodimers, such as AA and BB, or heterodimers, such as AB). The oestrogen receptor Encoded on the long arm of chromosome 6, the human oestrogen receptor (ER) is composed of 595 amino acids and exists as a 66 kDa protein in cells.12 The oestrogen receptor preserves the modular architecture demonstrated in other members of the nuclear receptor superfamily. The C-region or DNA-binding domain contains 100 amino acids with nine cysteines in ®xed positions containing two zinc ions, the zinc ®ngers. The

Hormonal treatment 473

speci®city of receptor binding to the hormone-responsive element of DNA resides within the zinc moieties of this domain. The interaction of the zinc ®ngers with DNA controls which genes will be regulated by the receptor. When the structure of the DNA binding domain was analysed crystolographically it was shown that the DNA-binding competent form of the receptor was a dimer and in each dimer the ®rst zinc ®nger of each monomer binds to the DNA target.9,13 This arrangement places each subunit into adjacent major grooves of the DNA double helix. The second zinc ®nger is not directly involved in DNA binding but is involved in the formation of contacts between receptor dimers. The hormone-binding domain, or E region, of the ER consists of 251 amino acids and is responsible for hormone binding and eventual dimerization. This region also contains the transcription activation function called TAF-2. TAF-2 depends on hormonal binding for full activity. The hormone binding domain contains a characteristic structural feature with helices that form a pocket.14 After hormonal binding, this pocket undergoes conformational changes that create new surfaces which can interact with adaptor proteins. Over 30 di€erent adaptor (coactivator or co-repressor) proteins have been identi®ed. The co-activator proteins have histone acetylase activity, whereas the co-repressors exhibit histone deactylation.15,16 Histones do more than pack the genome into the nucleus. Histone acetylation by co-activator proteins adds acetyl groups to lysine moieties, loosens histone binding to DNA, and exposes critical nucleotide regions to the transcriptional machinery leading to transcriptional activation and mRNA synthesis. Conversely, histone deacetylation terminates transcription by allowing DNA to wrap more tightly around the histone proteins. Recently, an alternative pathway of oestrogen receptor action has been reported involving DNA binding to a site other than the hormone responsive element.17,18 By this mechanism, the fos and jun proteins directly bind to their respective DNA recognition sites (AP-1), and are modulated either positively or negatively by the associated, ligand-bound and dimerized oestrogen receptor. Whereas the classical mechanism of oestrogen action involves the direct binding of receptor to DNA at the ERE, the alternative pathway involves protein-to-protein (receptor-jun/fos) interaction to exert control at the AP-1 DNA site. After cloning of the oestrogen receptor, there was general agreement that only one form of the oestrogen receptor existed. This contrasted with multiple isoforms of other members of the nuclear receptor superfamily. Surprisingly, a new oestrogen receptor, termed ERb, as opposed to the original oestrogen receptor, ERa, was subsequently identi®ed in cDNA libraries from human testis, a tissue not usually considered to be a major oestrogen target.19 The human ERb contains 530 amino acids and is a product of a di€erent gene on chromosome 14 q22-24 located at a site near the gene associated with early onset of Alzheimer's disease.20 Comparison of the two human oestrogen receptors shows a high degree of conservation in the DNA-binding and the ligand-binding domains, as opposed to considerable variation in the A/B, hinge and F domains (Table 1). The highest levels of ERb mRNA expression occur in the kidney, thymus and small intestine, with lower levels documented in lung, spleen, pituitary gland, leukocytes, bone marrow, colon and uterus.20 The results suggest that while some tissues may exclusively contain one isoform of the oestrogen receptor, other tissues contain both ERa and ERb. Both ERb and ERa form functional homodimers after binding to their ligand.21,22 The dimerized complex then binds to the hormone responsive element of DNA. The formation of ERa/ERb heterodimers has been reported which may further modulate

474 E. Podczaski and R. Mortel Table 1. The degree of homology between the two forms of the oestrogen receptor as determined by receptor domain.20 Homology between ERa and ERb (%) Regulatory domain DNA-binding domain Hinge region Ligand-binding domain F-region

17.5 97.0 30.0 59.1 17.9

oestrogen action. Tissues expressing both isoforms of the oestrogen receptor may contain target genes which are speci®cally activated by such heterodimers. Clinical use of progestins in endometrial cancers Primary progestin therapy for endometrial cancers Although standard therapy for endometrial cancer is hysterectomy and bilateral salpingo-oophorectomy with appropriate staging, selected patients have been treated with progestational agents as primary therapy in an attempt to preserve childbearing potential. Endometrial cancer is largely a disease of post-menopausal women. However, a subset of young women can present with this disease, often in a setting of obesity, irregular menses, chronic anovulation and the clinical stigmata of polycystic ovarian syndrome. This group of women poses a therapeutic dilemma. Standard therapy is incompatible with preservation of fertility, a major concern for these individuals. Farhi and colleagues described ten cases of endometrial cancer arising in women under the age of 25.23 The slides were reviewed by at least two pathologists in order to distinguish these cases from adenomatous/atypical hyperplasia. Seven of the ten patients demonstrated the clinical characteristics of women with polycystic ovarian syndrome. Nine of the tumours were well-di€erentiated endometrioid adenocarcinomas and were limited to the endometrium (Figure 4). In one case, a moderatelydi€erentiated adenosquamous carcinoma involved an ovary and the pelvic wall. In ®ve cases, progestins were used as primary therapy; three of the patients had no further evidence of tumour on repeat endometrial biopsy. One of the three patients successfully treated with progestins went on to deliver two term pregnancies. Kim and associates treated seven patients with progestins alone for grade I endometrial adenocarcinomas.24 The patients ranged from 19 to 41 years of age and initially received 160 megesterol acetate a day for 3 months. Four of the seven patients had an initial response to progestins with no residual disease detected on follow-up endometrial sampling. This series was then combined with 14 additional patients identi®ed by MEDLINE search. By the aggregate data, 13 of the 21 patients (62%) had an initial response to progestins. Six viable infants were delivered to three of the patients after use of progestins. In another recent review 12 patients under the age of 40 with grade I endometrial cancers were treated with high-dose progestins.25 Nine of the twelve progestin-treated patients had documented regression to benign endometrial ®ndings. Three of the patients with a response to progestins went on to a total of ®ve term pregnancies. Two additional case reports have also documented progestin-induced regression of grade I endometrial adenocarcinomas in patients of 35 years of age or

Hormonal treatment 475

Figure 4. Endometrial carcinoma in a 40-year-old woman with polycystic ovarian syndrome. The uterus was sectioned in a series of strips to demonstrate a non-invasive tumour limited to the corpus of the uterus. Myometrium (M) and arrows mark the intersection of the polypoid tumour contained in the endometrial cavity and the myometrium.

less.26,27 In both cases subsequent pregnancies occurred as a result of in vitro fertilization and embryo transfer. The data suggest that young patients with grade I endometrial adenocarcinomas can be treated with progestins alone. The majority (62±75%) of patients will have a response to such therapy as demonstrated by negative follow-up endometrial curettage.24,25 However, primary progestin therapy is not without some risk. Three of 13 patients with an initial response to progestins later developed recurrence of disease, and one of the three patients had metastatic disease.24 In this later case, progestins delayed de®nitive surgery and potentially adversely a€ected the prognosis. One of the nine progestinresponders reported by Randall and Kurman experienced a recurrence of the endometrial cancer.25 Another two patients with grade I cancers treated with progestins were found to have coexistent ovarian cancers after 3 and 6 months of hormonal therapy. Both patients had concomitant, stage I endometrioid adenocarcinomas arising in endometriosis. Although the optimal patient characteristics for primary progestin therapy are unknown, suitable candidates are young women with grade I tumours and no or minimal myometrial invasion. Early data suggest a 5% risk of disease progression during or after progestin therapy.24 Dilatation and curettage should be considered as part of the initial evaluation because it remains the standard against which other sampling methods are compared. Magnetic resonance imaging (MRI) may be useful in detecting deep myometrial invasion and metastasis. Judicious patient selection and meticulous follow-up are mandatory for primary progestin therapy. Such treatment is best reserved for young women desirous of fertility or debilitated patients who are not candidates for surgery or radiotherapy.

476 E. Podczaski and R. Mortel

Adjuvant progestin therapy for early endometrial cancers Several studies have considered the adjuvant use of progestins after surgical therapy of `early' endometrial cancers. Lewis and colleagues randomly assigned 285 patients to adjuvant depot medroxyprogesterone acetate (MPA) and 287 women to no further therapy after initial surgery or pre-operative radium followed by hysterectomy and bilateral salpingo-oophorectomy.28 After entry into the study, the extent of disease was evaluated after primary therapy. Patients with extra-uterine disease were excluded from the study, and only patients with disease limited to the uterine corpus were considered in the analysis. Interim assessment of data at 4 years failed to demonstrate a survival bene®t with the use of adjuvant progestins. Similar results were obtained in prospective randomized studies done in the United Kingdom by Macdonald and associates, as well as in a large multicentre trial by De Palo and colleagues.29,30 In a large prospective study by Vergote et al31, 1148 patients undergoing hysterectomy and bilateral salpingo-oophorectomy for a stage I or II endometrial cancer were randomly assigned to adjuvant progestins or no additional treatment. Patients with tumours extending into the outer third of the myometrium or grade III disease were treated with external beam radiotherapy to the pelvis. Stage II patients received pre-operative intracavitary radiotherapy. Progestin therapy consisted of a loading dose of 5 gm of 17-a-hydroxyprogesterone caproate, followed by bi-weekly injections of 1 gm for 1 year. After exclusion of 64 randomized patients from analysis, the duration of follow-up ranged from 42 to 132 (median of 72) months. There was no statistically signi®cant di€erence between both groups with regard to overall survival or relapse rate. Con¯icting results were reported by Urbanski and colleagues.32 In this prospective study, 205 patients were randomly assigned to adjuvant progestins (bi-weekly intramuscular injections of 17-a-hydroxyprogesterone caproate for 1 year) or no further therapy. Patients given progestins had a statistically signi®cant survival advantage as compared to those with no additional therapy. The study was limited by the relatively low numbers of patients in each arm, as well as study design. Thirty percent of the patients enrolled had stage II or III disease. Available clinical studies on the use of adjuvant progestins have obvious ¯aws. In the study reported by Lewis and colleagues28, the exclusion of 318 patients after randomization, the presence of multiple treatment arms and an end-point of 4 years of observation all mitigate against a meaningful analysis. Macdonald and associates29 also excluded 148 patients after randomization, leaving only 281 evaluable patients. The study of DePalo et al30 was also limited by the short period of follow-up (median 22 months). However, the preponderance of clinical studies have not demonstrated a signi®cant survival bene®t with adjuvant use of progestins in early endometrial cancer. The inability of relatively large randomized trials to demonstrate a survival advantage for adjuvant progestins re¯ects the fact that such treatment is most likely to bene®t those with well-di€erentiated, PR-positive tumours. This is also a group with a better prognosis than those individuals with poorly di€erentiated tumours, a cohort with a substantially higher recurrence rate. At present there appears to be no indication for post-operative progestins following surgical therapy of early endometrial cancers. Use of progestins for recurrent/advanced endometrial cancers Studies by Kelley and Baker demonstrated that progestational agents can be used to treat recurrent endometrial cancers with objective responses in approximately 30% of

Hormonal treatment 477

patients.2 However, carefully performed studies in the past two decades have reported substantially lower response rates. Piver and colleagues used MPA or 17-ahydroxyprogesterone caproate to treat 114 women with metastatic or recurrent endometrial carcinoma.33 The overall objective response rate was 15.8%, with 7% of the patients experiencing a complete response. Response rates were unrelated to the progestational agent used. Patients whose disease recurred 3 or more years after initial therapy had a signi®cantly greater increase in response to progestational agents as compared to those patients whose disease recurred earlier. In a later study by Podratz et al34, progestational agents produced an objective response rate of 11.2% among 155 patients with advanced or recurrent endometrial cancer. Overall survival after initiation of therapy was 40% at 1 year, 19% at 2 years, and 8% at 5 years. Survival was statistically related to the degree of tumour di€erentiation, the estimated tumour volume at the initiation of therapy, and the time interval from primary treatment to the start of hormonal therapy. Similar data were reported by Thigpen using data from the Gynecologic Oncology Group.35 Patients with advanced or recurrent endometrial cancer were given 150 mg of MPA orally a day. Of 331 patients with measurable disease, the response rate was 18%, with 10% of the patients experiencing a complete response. The route of administration, nature of the progestin, and dose intensi®cation did not appear to in¯uence either the response rate or survival in advanced or recurrent endometrial carcinomas. Sall and colleagues randomized 22 patients to either oral MPA, 50 mg three times a day by mouth, or intramuscular MPA (300 mg weekly).36 Serum levels were consistently higher in the group treated with oral MPA, demonstrating that adequate levels can be achieved with the oral route. In a retrospective analysis of several studies Kauppila concluded that the route of administration was of minor clinical signi®cance in the overall response of progestins.37 Furthermore, in two retrospective studies the therapeutic response observed was independent of the progestational agent selected.33,34 Dose intensi®cation also appears to o€er limited therapeutic bene®t. In a randomized, Gynecological Oncology Group trial of 200 mg versus 1000 mg of MPA by mouth, there was no advantage to the higher-dose schedule.38 Using high-dose megesterol acetate (800 mg by mouth per day) in advanced or recurrent endometrial cancers, Lentz and colleagues obtained a 24% overall response rate with a complete response rate of 11%.39 High-dose megesterol acetate had activity in endometrial carcinoma but did not appear to o€er an advantage over lower-dose progestins.

Predicting a response to progestin therapy in advanced/recurrent disease Measurement of receptor concentrations in endometrial cancers Receptor concentrations can be determined quantitatively by biochemical ligand binding analysis and immunoassay, or qualitatively by immunohistochemistry. The most commonly used biochemical ligand assay is the dextran-coated charcoal (DCC) analysis in which a tissue homogenate is prepared and the supernatant (cytosol) is incubated with increasing concentrations of a radioactively labelled steroid. With the development of monoclonal antibodies against epitopes of the ER and PR molecules, a quantitative, solid-phase enzyme immunoassay has become available for determination of receptor concentrations. Receptors in tissue cytosol bind to polystyrene beads coated with a monoclonal antibody raised to the receptor. A second monoclonal antibody conjugated with peroxidase is later incubated with the receptor±bead complex. The amount of

478 E. Podczaski and R. Mortel

bound receptor is assessed by a colorimetric reaction mediated by the bead-bound peroxidase. Monoclonal antibodies raised against the ER and PR are also utilized in qualitative immunohistochemical assays in frozen or paran-embedded tissue sections. An estimate of the distribution and cellular localization of the receptors can be made, along with a semi-quantitative evaluation of receptors based on the intensity of immunostaining and the fraction of staining tissue components. Such methods have con®rmed the nuclear localization of both the oestrogen and progesterone receptors. Numerous studies have reported on oestrogen and progesterone concentrations in endometrial cancers.40±42 Measurements of receptor concentrations have shown considerable variation. The choice of assay method, methodological di€erences and the criteria for a positive determination have all contributed to this variation. Despite these di€erences, a general pattern of receptor levels in endometrial cancers has emerged. Essentially, all endometrial cancers are ER-positive, and the oestrogen receptor concentrations are similar to those seen in the late proliferative phase of normal endometrium. The values for progesterone receptor concentrations are relatively low and comparable to those obtained in normal secretory endometrium. The relationship between steroid receptor concentrations and tumour grade has been extensively studied.43±46 Although no de®nite pattern in oestrogen receptor levels exists, the progesterone receptor concentrations vary with the degree of tumour di€erentiation. Well-di€erentiated adenocarcinomas contain high concentrations of progesterone receptors with a progressive decrease in ER-levels in more anaplastic tumours. Aggressive histological variants, such as clear-cell carcinomas, demonstrate signi®cantly lower levels of ER and PR as compared to typical endometrioid adenocarcinomas.47 Although there is general agreement about the relationship between progesterone receptor levels and the histological di€erentiation of the tumour, the correlation is far from perfect. Some highly di€erentiated adenocarcinomas with low PR levels and poorly di€erentiated cancers with signi®cant receptor concentrations have been reported. Analogous to the clinical management of breast cancer, the receptor status of recurrent endometrial carcinoma was also evaluated as a predictor of hormone responsiveness. In a prospective study, Benraad and colleagues obtained biopsy specimens in 150 patients with untreated endometrial cancer.48 Thirteen patients eventually developed recurrent (metastatic) disease and received progestins. Five of the patients were ER- and PR-negative and were refractory to progestin therapy. Ehrlich and associates also observed a statistically signi®cant relationship between the presence of the progesterone receptor and responses to progestins in advanced and recurrent endometrial cancer.49 A pooling of results from several studies demonstrates the response of advanced or recurrent endometrial adenocarcinomas to progestational agents as categorized by PR-levels.45,46,48,49 Although a signi®cantly higher response rate is observed in patients with PR-positive tumours, a variable response is found in both the receptor-positive and receptor-negative groups. The complexity of tissue and tumour heterogeneity may be the underlying reason for such discordant results. Major factors complicating attempts to develop prognostic tests for the progestin sensitivity of endometrial carcinoma include the risk of specimen contamination by adjacent tissues and the multiple levels of tumour heterogeneity. In most studies reported, steroid receptor measurements are performed on homogenates of tissue specimens obtained at endometrial sampling or hysterectomy. Normal tissue, hyperplastic endometrium and carcinoma all contribute variably to the receptor levels of the sample. Progesterone receptor levels assessed by immunohistochemistry were

Hormonal treatment 479

compared with receptor concentrations obtained by the dextran-coated charcoal method in 24 primary endometrial cancers.50 Comparison of the two methods showed good correlation between the results with a concordance of 83% (20 of 24 cases). The discordant observations were due in part to lack of immunohistochemical staining for progesterone receptor in neoplastic cells, but low or moderate nuclear staining of adjacent benign tissues. Tumour heterogeneity has also been evaluated using monoclonal antibodies raised against the progesterone receptor.50 Using the conventional architectural and cytologic criteria of di€erentiation, it is not uncommon to observe subpopulations of neoplastic cells with varying degrees of di€erentiation within the same tumour. Furthermore, the proportion of positive cells stained for the progesterone receptor and the intensity of the reaction were highly variable within each PR-positive tumour. Each of the positive tumours demonstrated marked staining variability in di€erent microscopic ®elds, as well as adjacent glands and cells within the same neoplastic gland. Under such circumstances, it is unreliable to predict which tumour cell population will predominantly contribute to the biological behaviour of an eventual recurrence. Use of tamoxifen in endometrial cancers Tamoxifen, SERMs, and their mechanism of action Tamoxifen is a triphenylethylene derivative similar to diethylstilbestrol. The citrate salt of the trans isomer is used clinically because of its increased anity for the oestrogen receptor. The customary dosage is 20 mg a day, and the drug is readily absorbed after oral administration. Serum half-lives of tamoxifen and its metabolites range from 7 to 14 days, allowing once-daily administration.51 Although regarded as an `anti-oestrogen' in breast tissue, it is now known that tamoxifen actually has oestrogenic or agonist activity at other sites. Tamoxifen treatment of post-menopausal women results in preservation of bone mineral density, reduction in LDL-cholesterol and an increase in sex-hormone binding globulin, responses typically associated with the use of an oestrogen agonist.52,53 Furthermore, patients treated with tamoxifen for breast cancer may experience an increased risk of endometrial hyperplasia or carcinoma as a result of oestrogen agonist activity at the level of the endometrium.54,55 The `anti-oestrogen' tamoxifen is, in reality, the ®rst selective oestrogen receptor modulator (SERM), a new category of therapeutic agents which mimic the e€ect of oestrogens in some tissues, but act as oestrogen antagonists in others. All SERMs act by binding with high anity to the oestrogen receptor, and yet produce tissue- and drug-speci®c responses. Such data result in a paradox which cannot be explained in the context of existing models of oestrogen receptor function. A possible explanation for the tissue-speci®c responses to di€erent oestrogen receptor-ligands is that they interact with di€erent receptors (ERa or ERb), each of which may be di€erentially expressed in target tissues. The overall biological response to a drug re¯ects the sum of its relative agonist/antagonist activities with the two di€erent receptors. Another means of modulating the response to a particular oestrogen receptor±ligand complex is variable binding at the DNA AP-1 site, which is mediated by fos and jun. Recent evidence suggests that tamoxifen binding to ERb results in transcriptional activation via the AP-1 site, whereas the use of oestradiol as the ERb-ligand causes transcriptional suppression.56

480 E. Podczaski and R. Mortel

The existence of two oestrogen receptors does not appear to explain suciently the complex pharmacology of SERMs. Individual members of this class of drugs can demonstrate di€erent biological activity even in tissues expressing only a single isoform of the oestrogen receptor. Using protease digestion assay and analysis of crystal structures, it has been demonstrated that the ER, when bound to tamoxifen, raloxifene or other SERMs, can adopt conformations which are intermediate between that induced by oestradiol and that of the unbound receptor.57,58 Thus, in addition to the `on' and `o€' con®gurations of the oestrogen receptor proposed by traditional models, intermediate conformations are possible, each of which is associated with a di€erent spectrum of ER agonist/antagonist activities. A major determinant of SERM activity is the ability of the target cell to distinguish between receptor con®gurations induced by binding of drug to the oestrogen receptor. The molecular basis for this discrimination appears to reside in adaptor proteins which are di€erentially expressed in various target tissues. So far over 30 di€erent adaptor (co-activator, as well as co-repressor) proteins have been identi®ed. These proteins participate in the process whereby the receptor contacts the DNA-hormone responsive element and transcription is initiated.59 Overexpression of the SRC-1 (steroid receptor co-activator-1) protein potentiates the transcriptional activity of estradiol activated ER and also imparts agonist activity to the tamoxifen bound ER.60 Conversely, the introduction of L7/SPA, another adaptor protein, into cultured breast cancer cells, converted tamoxifen from an agonist to an antagonist.61 Clinical use of tamoxifen in recurrent/advanced endometrial cancer Based on prior experience with breast cancer, tamoxifen, either as a single agent or in combination with other hormones, has been used in the treatment of advanced and recurrent endometrial carcinoma. These studies were initiated at a time the agonist action of tamoxifen in the endometrium was not yet appreciated. Reviewing eight studies using tamoxifen (20 to 40 mg per day), Moore and associates obtained a pooled response rate of 22%.62 Edmunson and colleagues, using 10 mg tamoxifen twice daily, noted an overall response rate of 21% in patients not previously treated with progestational agents.63 However, none of the 22 patients previously treated with progestins responded to tamoxifen. Similar results were obtained from a Gynecologic Oncology Group study.64 None of the 19 patients with advanced or recurrent endometrial carcinoma had a response to tamoxifen after resistance to hormonal treatment with progestins. As a result, it is unlikely that patients with advanced or recurrent endometrial cancer unresponsive to progestational agents will experience a response to tamoxifen as a single agent. Tamoxifen and progestin in recurrent/advanced endometrial cancer The information that tamoxifen augmented progesterone receptor concentrations in endometrial carcinoma led to the prediction that pre-treatment with tamoxifen may potentiate the degree and duration of response of these tumours to progestin therapy. Oestrogen and progesterone receptor concentrations were measured in tumours from 25 patients with previously untreated endometrial cancers before and after a course of tamoxifen.65 On initial biopsy 52% of the tumours were progesterone receptor-positive, whereas 84% were receptor-positive after use of tamoxifen. The increased incidence of measurable progesterone receptors with use of tamoxifen was predominantly observed in tumours with grade I and II histology. Carlson and colleagues used both tamoxifen

Hormonal treatment 481

and progestins to treat 12 patients with metastatic endometrial cancer.65 All patients were post-menopausal and none had previously received hormonal therapy. One patient had a complete response, three had a partial response (33% overall response rate), and two had stable disease. In a similar study using tamoxifen and megesterol, a 19% response rate was observed in 42 patients suitable for evaluation.66 Kline and associates treated 20 consecutive patients with poorly di€erentiated recurrent or metastatic endometrial cancers with sequential courses of tamoxifen and MPA.67 Treatment consisted of tamoxifen (10 mg twice daily) for 5 days followed by MPA (50 mg twice daily) on days 6 through 25. The treatment cycle was repeated after a 5-day rest. Only one patient with pulmonary metastasis and extra-uterine pelvic disease had a complete response to treatment. Tatman and colleagues treated 15 patients with advanced endometrial cancer with sequential ethinyl oestradiol (50 mg a day for days 1± 7) and MPA (400 mg daily on days 8±25).68 All patients were post-menopausal and had grade II or III disease. There were no signi®cant clinical responses in 12 evaluable patients. Four of the 15 patients treated experienced thromboembolic complications. The Gynecologic Oncology Group has completed a phase II study of alternating courses of megesterol and tamoxifen. Patients with histologically documented persistent or recurrent endometrial carcinoma were given a 3-week course of megesterol (160 mg a day) followed by 3 weeks of 40 mg tamoxifen a day. Preliminary results of the study are still pending.

GnRH analogues in recurrent/advanced endometrial cancer The hormonal therapy of advanced or metastatic endometrial adenocarcinoma also includes the potential use of gonadotropin-releasing hormone (GnRH) analogues. These compounds have been clinically useful in other hormonally dependent neoplasms such as prostate and breast cancer. GnRH analogues cause an initial rise followed by suppression of pituitary gonadotropins and a resulting fall in sex steroids. GnRH analogues may also act via speci®c tissue receptors. Emons and colleagues suggested that the GnRH antagonist SB-75 directly inhibited the proliferation of human endometrial cancer cell lines in vitro, possibly by combining with high-anity binding sites.69 Using a similar in vitro model, Kleinman and coworkers demonstrated growth inhibition without any change in cell cycle parameters, presumably by the induction of programmed cell death or apoptosis.70 Clinical data on the use of GnRH analogues in patients with advanced endometrial cancer are limited and con¯icting. Using leuprolide or goserelin, Gallagher and associates treated 17 patients with recurrent endometrial cancer.71 Fourteen of the patients had been previously treated with progestins, either in an adjuvant setting or as prior treatment of recurrent disease. Six of the seventeen patients (35%) experienced a complete or partial remission which continued for a median of 20 months. Jeyarajah and colleagues, using the same agents, treated 32 consecutive patients with recurrent endometrial carcinoma.72 Nineteen patients had been previously treated with MPA and another four received tamoxifen. An objective response was seen in nine patients (two complete responses and seven partial responses). Two subsequent reports were less favourable. Of 34 patients with advanced or recurrent endometrial carcinoma, there were no responders to leupriolide.73,74 Nine of the patients received prior progestational agents. Although the drug was well tolerated, leuprolide did not appear to be an active drug in advanced endometrial cancer. The Gynecologic Oncology Group has recently completed a phase II study of

482 E. Podczaski and R. Mortel

goserelin in patients with recurrent or persistent endometrial cancer. Although the protocol has been completed, preliminary results are not yet available.

Combined hormonal and cytotoxic chemotherapy for recurrent/advanced endometrial cancer Over the past two decades there have been at least 10 reports (Table 2) describing the use of hormones and cytotoxic chemotherapy for advanced and recurrent endometrial cancer.75±84 Although the majority of the reports describe small series of patients, two studies contained more than 100 patients. Most studies have used megesterol acetate or MPA in combination with adriamycin chemotherapy. Three of the reports also include the use of tamoxifen in the hormonal regimen. Most response rates obtained were in the 33 to 60% range. Although the addition of hormones may impart some survival advantage, systematic phase III trials demonstrating such a bene®t have yet to be done. Table 2. Ten studies over the past two decades using combined hormonal and cytotoxic chemotherapy for advanced and recurrent endometrial carcinoma. The table summarizes agents used, number of evaluable patients and response rates. Tam ˆ tamoxifen. Number of patients evaluated

Response rate (%)

Reference

Hormone

Cytotoxic agents used

75

Megestrol

5-FU, Cyclophosphamide, Doxorubicin

29

45

76

Megestrol

5-FU, Cyclophosphamide, Doxorubicin

111

22

77

Megestrol Megestrol

5-FU, Melphalan 5-FU, Cyclophsophamide Doxorubicin

77

78

Megestrol

Cyclophosphamide, Cisplatin, Doxorubicin

15

60

79

Megestrol + Tam

5-FU, Melphalan

50

48

80

MPA/Tam

5-FU, Cyclophosphamide, Doxorubicin

23

43

81

Megestrol

Cyclophosphamide, Cisplatin, Doxorubicin

15

33

82

MPA

Cyclophsophamide, Cisplatin, Doxorubicin

15

53

83

Megestrol

Cisplatin, VP-16, Doxorubicin

50

54

84

Megestrol ‡ Tam

Carboplatinum

13

77

78

36.8

THE FUTURE From a receptor perspective, there are many similarities between cancers of the breast and endometrium. Both arise as carcinomas from a hormonally responsive epithelium. Either disease can be classi®ed by the presence or absence of the oestrogen and

Hormonal treatment 483

progesterone receptor, and such information has prognostic implications. Anaplastic tumours are often characterized by the failure to express ER and PR, whereas biologically less aggressive disease is often receptor-positive. Furthermore, receptorpositive disease can be treated by hormonal manipulations which may eventually lose ecacy. Resistance to endocrine therapy remains a continuing problem in both diseases. The following is a brief synopsis of receptor studies in breast cancer. Many of these same steps will need to be retraced in investigating a similar problem in endometrial cancer. As the loss of the ER gene in primary breast cancer does not account for the vast majority of ER-negative breast carcinomas, another possibility is lack of gene expression. Carmeci and colleagues quanti®ed the levels of RNA found in archival breast cancer specimens by reverse transcriptase-polymerase chain reaction.85 Of 31 ERnegative tumours, nine had low levels of ER mRNA and 21 had medium levels of ER mRNA expression. The authors suggested that ER expression was controlled at the level of transcription in approximately 30% of ER-negative breast carcinomas. Possible explanations include proposed altered enhancer sequences a€ecting control of ER gene expression and aberrant methylation of the ER gene. Another consideration is the loss of ER and PR function in receptor-positive breast tumours. Loss of receptor function can occur by a variety of di€erent mechanisms including point mutations of the gene, alternative mRNA splicing, altered phosphorylation of the receptor protein, and modi®ed interactions with the adaptor (co-activator or co-repressor) proteins. Mutations have been detected in the E domain altering hormonal binding and response to ligands. Variant ER proteins can also arise as a result of splicing. These variant ER proteins lack speci®c functional domains and may alter the response to oestrogens and oestrogen antagonists. van Dijk and colleagues have studied di€erences in the prevalence and functional activity of ERa variant mRNAs in 21 normal portions of breast tissue and 41 primary breast cancers.86 The presence of the wild-type ER relative to the total amount of ER di€ered signi®cantly between the normal and cancerous tissue examined. Furthermore, the ER variants with altered function present in normal breast tissue mainly re¯ected splicing variants in which one exon was deleted, whereas in breast cancers only half of all variants lacked just one exon. The receptor issue germane to breast cancer is equally relevant to endometrial cancer. The hormonal treatment of endometrial cancers depends on receptor expression. Lack of the progesterone receptor removed the `handle' by which these tumours can be manipulated. E€orts have been directed at inducing PR expression using compounds with oestrogen agonism, such as tamoxifen. Tamoxifen therapy can induce PR synthesis even in tumours with low initial levels of progesterone receptors, making such tumours potentially responsive to progestin therapy. However, such an e€ect is most pronounced in tumours with favourable clinicopathological characteristics.87 Using a nude mouse model, combination therapy with tamoxifen and progestins was e€ective in arresting the growth of transplanted human endometrial cancers.88 However, after a 15 to 20 week tumouristatic period, the tumours began to regrow, reminiscent of the clinical situation. Lack of progestin sensitivity during the period to tumour regrowth appeared to re¯ect the absence of the progesterone receptor. Recently, the Gynecologic Oncology Group has completed a study to evaluate the ecacy of tamoxifen (40 mg daily) plus intermittent administration of MPA (200 mg per day on alternate weeks) as treatment of advanced, recurrent, or metastatic endometrial cancer. All patients had histologically con®rmed disease, and all grades of disease were eligible for study entry. The results of the protocol should be available in the near

484 E. Podczaski and R. Mortel

future. Further studies will be necessary to determine whether better response rates can be achieved with other dosage and schedule regimens of tamoxifen and progestins. Despite e€orts to modulate the expression of progesterone receptors, future e€orts will need to determine the mechanisms responsible for the phenotypic lack of PR observed in many endometrial carcinomas. Grade I tumours are often PR-positive; however, these tumours are less likely to recur after therapy. Unfortunately, it is the poorly-di€erentiated, hormone receptor-negative tumours that are more likely to recur, and these tumours are usually refractory to hormonal therapy. Insights into the genetic mechanisms responsible for the absence of PR expression/ function are basic to any e€ort at endocrine therapy in advanced and recurrent endometrial cancers. Early reports dealing with molecular alterations in the PR gene and its transcription have appeared in the literature. Assikis and colleagues performed single-stranded conformational polymorphism analysis (SSCP) on 35 endometrial cancers.89 Four point mutations were identi®ed in three patients with grade III carcinomas. Kohler and associates performed PCR and SSCP of the entire coding region of the ER gene on genomic DNA extracted from 56 snap frozen endometrial cancers.90 Although seven cancers with mobility shifts were identi®ed, in six of the seven cases the alterations were consistent with infrequent silent polymorphisms. In the seventh case, the sequence alteration was a somatic missense mutation at codon 537 in the region of the ER gene encoding the hormone binding domain of the receptor protein. As a result the authors felt that such infrequent DNA mutations in the ER gene were unlikely to account for the variation in oestrogen receptor expression observed in endometrial cancers. As in other cancers, basic work will need to determine the genetic and molecular basis for receptor expression. Only such fundamental insights will provide future direction in the endocrine therapy of advanced and recurrent endometrial cancers. CONCLUSIONS Although initial studies on the use of progestins in metastatic or advanced endometrial carcinoma reported response rates of approximately 30%, carefully performed studies in the past two decades have reported substantially lower response rates. Patients with well to moderately di€erentiated, PR-positive tumours are more likely to respond to such hormonal therapy. Unfortunately, the poorly di€erentiated tumours are more likely to recur. These tumours are usually PR-negative and refractory to progestins. Although some advanced tumours also respond to tamoxifen, patients refractory to progestins are usually also unresponsive to tamoxifen. Tamoxifen and progestins have been used together to treat advanced endometrial carcinomas. Theoretically, tamoxifen may enhance progesterone receptor expression and increase the response rate to progestins. However, preliminary clinical data are scanty. Furthermore, initial studies have indicated that such therapy may be most ecacious in tumours of better di€erentiation. Progestins have been used in the primary therapy of young women with welldi€erentiated adenocarcinomas. Such women usually have the clinical stigmata of chronic anovulation, obesity and the polycystic ovarian syndrome. Judicious patient selection and meticulous follow-up are mandatory for such primary progestin therapy. Progestins have also been used in an adjuvant setting as treatment following de®nitive surgery for endometrial cancers. A number of relatively large randomized trials have failed to demonstrate a bene®t for such treatment. At present there appears to be no

Hormonal treatment 485

Practice points . aims: use and limitations of hormonal therapy in primary and metastatic/ recurrent endometrial cancers, as well as use of such treatment in an adjuvant setting . management: response to progestational agents re¯ects PR status, which, in turn, is related to histology; response rates to progestins in the treatment of advanced/recurrent endometrial cancer are approximately 15%; response rates to progestins are una€ected by route of administration and dose intensi®cation; progestins are not useful in adjuvant treatment of early endometrial cancers following surgery; tamoxifen is ine€ective in tumours refractory to progestins; data regarding use of GnRH analogues are con¯icting . investigation: use of PR assays to assess potential responses to endocrine therapy; PR levels related to tumour histology

Research agenda . mechanism responsible for suppression of PR expression in cancers arising from hormonally responsive tissues . modulation of PR expression by use of SERMs indication for post-operative progestins following the surgical therapy of early endometrial cancer. Despite past e€orts to modulate the expression of progesterone receptors, future e€orts will need to determine the mechanisms responsible for the phenotypic lack of PR expression in many endometrial cancers. This is a problem common to the clinical treatment of breast cancers, as well. Lack of receptor expression severely limits the clinical usefulness of endocrine therapy in both of these diseases.

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